Báo cáo khoa học: Multidrug efflux pumps: drug binding – gates or cavity? Emily Crowley and Richard Callaghan pdf

MINIREVIEW
Multidrug efflux pumps: drug binding – gates or cavity?
Emily Crowley and Richard Callaghan
Nuffield Department of Clinical Laboratory Sciences, University of Oxford, UK
Properties of drug-binding sites in
ABCB1
P-Glycoprotein (also known as ABCB1, mdr1) has
long been associated with drug resistance in many
cancer types. This protein is a member of the ATP-
binding cassette (ABC) family and is believed to confer
drug resistance in cancer cells by mediating the active
outward efflux of chemotherapeutic drugs. Providing
information on the nature of the drug–ABCB1 interac-
tion has been a vital and synergistic pursuit alongside
efforts to locate the drug-binding sites on the protein.
The range and number of compounds ‘recognized’ by
ABCB1 are astounding and there remains no conclu-
sive explanation for this polyspecific behaviour.
Numerous pharmacological studies utilizing equilib-
rium drug binding, hydrolysis of ATP and steady-state
accumulation assays have demonstrated that ABCB1
contains multiple sites for drug interaction. Moreover,
complex allosteric interactions between sets of drugs
have been demonstrated which may involve negative
heterotrophy or conversely, permit the simultaneous
binding of two drugs [1–4]. ABCB1 displays a complex
mechanism of drug translocation across the membrane
that requires coupling between the energy-providing
nucleotide-binding domains (NBDs) and the trans-
membrane domains (TMDs), which contain the drug-
binding sites and the translocation conduit. There is

considerable debate concerning the characteristics of
coupling, specifically whether one or two ATP mole-
cules are hydrolysed per transport event and whether
ATP binding per se or the hydrolytic step is responsi-
ble for switching the binding site orientation during
Keywords
central cavity; coupling; domain interface;
drug binding; drug resistance; drug
transport; interdomain communication;
membrane protein; multidrug binding;
P-glycoprotein
Correspondence
R. Callaghan, Nuffield Department of Clinical
Laboratory Sciences, University of Oxford,
Level 4, Academic Block, John Radcliffe
Hospital, Headley Way, Oxford OX3 9DU,
UK
Fax: +44 1865 221 834
Tel: +44 1865 221110
E-mail:
(Received 22 July 2009, revised 1 October
2009, accepted 5 November 2009)
doi:10.1111/j.1742-4658.2009.07484.x
The role of the ATP-binding cassette ABCB1 in mediating the resistance to
chemotherapy in many forms of cancer has been well established. The pro-
tein is also endogenously expressed in numerous barrier and excretory
tissues, thereby regulating or impacting on drug pharmacokinetic profiles.
Given these prominent roles in health and disease, a great deal of biochem-
ical, structural and pharmacological research has been directed towards
modulating its activity. Despite the effort, only a small handful of com-

pounds have reached the later stages of clinical trials. What is responsible
for this poor return on the heavy research investment? Perhaps the most
significant factor is the lack of information on the location, physical fea-
tures and chemical properties of the drug-binding site(s) in ABCB1. This
minireview outlines the various strategies and outcomes of research efforts
to pin-point the sites of interaction. The data may be assimilated into two
working hypotheses to describe drug binding to ABCB1; (a) the central
cavity and the (b) domain interface models.
Abbreviations
ABC, ATP-binding cassette; ABCB1, the multidrug resistance P-glycoprotein; IAAP, iodoarylazidoprazosin; NBD, nucleotide-binding domain;
TM, transmembrane; TMD, transmembrane domain.
530 FEBS Journal 277 (2010) 530–539 ª 2009 The Authors Journal compilation ª 2009 FEBS
transport. Binding events are also not equal because
some interaction of certain compounds leads to trans-
port and the stimulation of hydrolysis, whereas others
are not transported and cause potent inhibition of
ATPase activity. Elucidating the location and biophysi-
cal properties of the drug-binding sites would provide
considerable input into the molecular interaction
between substrates ⁄ inhibitors with ABCB1. Structural
and pharmacological information of this ilk would facil-
itate the development of potent inhibitors of ABCB1 to
restore the effectiveness of many anticancer agents.
Strategies to locate the drug-binding
sites
The inability to precisely localize the drug-binding sites
on ABCB1 has not been caused by a lack of effort.
Since 1989, a large number of research teams have
employed numerous strategies to elucidate the precise
location of drug-binding sites on ABCB1 and a sum-

mary of the major findings is presented in Table 1.
This not an exhaustive list, but does highlight many
significant observations during the past two decades.
Initial attempts made use of photo-active versions of
ABCB1 inhibitors including [
125
I]- iodoarylazidoprazo-
sin (IAAP) and [
3
H]-azidopine [5–7]. Typically, the
drug was covalently attached to the protein, which was
then subjected to either protease digestion or chemical
cleavage. Protein fragments containing bound drug
were analysed using specifically generated antibodies to
identify the binding site. A more recent investigation
employed a similar strategy using a propafenone deriv-
ative to probe drug interaction [8]. The protein was
chemically cleaved and the fragments analysed by
MALDI-TOF MS. This enabled precise identification
of the fragments labelled by drug and enabled relative
quantitation of the amount of drug bound.
Resistant cell lines expressing ABCB1 may be gener-
ated by the long-term exposure of cells to anticancer
drugs. In the presence of high drug concentrations, this
strategy frequently produced mutations within the pro-
tein, many concentrated in the TMDs [9]. Mutated
forms of ABCB1 conferred a distinct resistance profile
to the wild-type protein, which was thought to reflect
alterations in drug binding or transport. Subsequent,
more sophisticated, studies used directed mutagenesis

to introduce mutations into targeted regions of the
protein [10–12]. The functional consequences of the
mutations were assessed using a range of assays includ-
ing the ability of ABCB1 to confer cellular resistance,
reduce intracellular drug accumulation, bind drug
and ⁄ or display drug-stimulated ATPase activity.
Frequently, a range of these assays was employed to
provide a more detailed understanding of the contribu-
tion of specific residues to protein activity. A popular
approach involved the mutagenesis of target residues
to cysteine, which enables conjugation of thiol-reactive
drug derivatives and exploration of the local environ-
ment and topography [13–16].
The current structural resolution of ABCB1 does not
provide atomic details on the drug-binding site. How-
ever, structural information is available for a number of
bacterial ABC transporters including one (Sav1866) that
is likely to act as a multidrug efflux pump [17–19]. Con-
sequently, a number of in silico molecular models of
ABCB1 have been developed based on these structures.
Several teams have attempted to ‘dock’ drugs to the
structure in the hope of identifying likely regions
involved in the drug–protein interaction.
What regions are implicated in binding?
This minreview is not intended to provide an extensive
description of the past two decades’ research into the
drug-binding sites of ABCB1. Overall, the studies high-
lighted in Table 1 can be divided into two broad
descriptions of the drug-binding sites: the central
cavity model and the domain interface model.

The central cavity model
The ongoing electron microcopy–structure investiga-
tions were the first to reveal that ABCB1 has a central
cavity which is likely to be aqueous filled and that the
TMDs have regions of discontinuity that may allow
access to the lipid milieu [20–24]. A number of bio-
chemical studies suggest that the transmembrane (TM)
helices lining this central cavity contribute to drug
binding [25].
The absence of a high-resolution structure (i.e. at or
better than 3 A
˚
) hampers the assignment of specific
helices and their constitutive residues involved in lining
the central pore. A series of investigations has
attempted to produce a topographical map of the cen-
tral cavity by covalent cross-linking of cysteines intro-
duced into prospective helices [13,26,27]. Cross-linking
agents of different lengths have been used to generate
a dimensional aspect to the map. Interestingly, forma-
tion of several of the cross-links is perturbed by the
presence of substrates or inhibitors of ABCB1; this has
been suggested to demonstrate that the residues impli-
cated in cross-link formation mediate drug interac-
tions. Moreover, the ability to form long-distance
cross-links has also been interpreted as evidence that
the residues are located on the helical face in contact
with the central cavity.
E. Crowley and R. Callaghan Drug binding – gates or cavity?
FEBS Journal 277 (2010) 530–539 ª 2009 The Authors Journal compilation ª 2009 FEBS 531

Table 1. Time-line of the search for the location of drug-binding sites on P-glycoprotein. The time-line contains a summary of the strategies
and observations obtained from studies specifically aimed at locating the sites of drug binding to P-glycoprotein. The time-line shows a
selection of the major advances in this area and does not include reference to numerous studies describing the nature or physicochemical
properties of the binding sites. DBS, drug binding site; EC, extracellular; IAAP, iodoarylazidoprazosin; NBD, nucleotide-binding domain; TMD,
transmembrane domain.
Date Reference Comments
1989 [5] Strategy: Photoaffinity labelling and protein digestion
Result: Two sites or one with components in each half
1990-1991 [7,52] Strategy: Labelling, digestion and antibody-mediated identification
Result: 6 kDa fragment labelled within ⁄ close to TM11 and TM12
1993 [10] Strategy: Phe fi Ser mutations within TM11 followed by transport ⁄ cytotoxicity assays
Result: TM11 contributes to DBS
1993 [6] Strategy: Labelling, digestion and antibody-mediated identification
Result: Labelling at two regions C-terminal to TM6 ⁄ 12
1993 [11] Strategy: Phe fi Ala mutations at nucleotides 335 (TM6) and 978 (TM12)
Result: Mutations altered resistance profile, suggesting that TM6 ⁄ 12 contributes to binding and ⁄ or
translocation
1994 [53] Strategy: Theoretical – molecular simulations
Result: Proposed that drugs intercalate between multiple Phe residues. Several helices contain Phe
residues which shields drug from the aqueous environment – implicate TM3, TM5, TM8 and TM11
forming DBS
1995 [54] Strategy: ABCB1 chimera assessed by cytotoxicity and photolabelling
Result: Loop between TM11 and TM12 (EC0) modulates resistance spectrum & may be involved in
translocation pathway
1997 [12] Strategy: Site-directed mutagenesis and cytotoxicity
Result: Mutations in TM6 alter the ability of cyclosporin A (not verapamil) to overcome resistance; TM6
involved in selectivity
1997 [55] Strategy: Photolabelling and protein digestion
Result: Differential effects of flupentixol on two [
125

I]-IAAP labelling sites – nonidentical binding sites in
N- & C-termini
1998 [56] Strategy: Chemical structure–activity relationships for substrates
Result: H-bonding patterns in substrates are key elements in drug recognition
1998 [57] Strategy: Site-directed mutagenesis of TM12
Result: N-terminal region of TM12 influences transport specificity
2001 [58] Strategy: IAAP labelling and chemical cleavage
Result: Three regions of labelling found; TM4–5, TM7–8 and post-NBD2. Single site for IAAP comprising
multiple spatial elements
2001 [59] Strategy: Effects of TM9 mutations on cytotoxicity and photolabelling
Result: Mutations in TM9 produce a distinct resistance pattern similar to TM6. TM9 and TM6 may
co-operate in IAAP labelling
2005 [8] Ligand:[
3
H]propafenone and analogues
Strategy: Photolabelling and identification with MALDI-TOF MS
Result: Two binding regions – TM3 ⁄ 11 and TM5 ⁄ 8. MsbA-based model suggest the two sites are at
TMD : TMD interfacial regions
2005 [2,60] Strategy: Mapping R ⁄ H sites; fluorescence approach
Result: H-site within bilayer leaflet region of ABCB1, whereas the R-site is in the cytosolic region. The
R-site can bind two drugs simultaneously
2005 [61] Strategy: Review of their site-directed mutagenesis studies
Result: Suggest a common drug-binding site in the central cavity Interfaces at TM5 ⁄ 8 and TM2 ⁄ 11 form
gates to the cavity and drugs negotiate passage through these gates
2006 [36] Strategy: Theoretical study – MsbA-based ABCB1 model
Result: Propose a large central binding cavity with a lateral opening to lipid bilayer. Cavity helices include
TM4, TM5, TM6, TM10, TM11 and TM12
2006 [62,63] Strategy: Directed mutagenesis and drug labelling
Result: Two studies suggesting that TM1 ⁄ 7 also contribute to the binding pocket in the central cavity
2006 [64] Strategy: Simulations to probe drug–ABCB1 interaction

Result: Argue that those residues at the interfacial region and that this is in contact with the polar head
group region of the membrane
2007 [35] Strategy: Sav186-based model used to characterize drug binding
Result: Proposed several key residues from TM1, TM5 and TM6 in drug binding
Drug binding – gates or cavity? E. Crowley and R. Callaghan
532 FEBS Journal 277 (2010) 530–539 ª 2009 The Authors Journal compilation ª 2009 FEBS
Mutations of several residues suggested to line the
central cavity have been independently shown to alter
ABCB1 function. Perhaps the most frequent reported
manifestation is an alteration in the cytotoxicity profile
– for example, increased resistance conferred to actino-
mycin D [9]. A change in the resistance profile may
indicate that the residue in question favours interaction
with one drug over another. Other reported manifesta-
tions are altered steady-state accumulation of ABCB1
substrates within cells and modified stimulation of the
basal rate of ATP hydrolysis. These alterations in
ABCB1 function have also been interpreted in terms
of the initial drug–protein binding event.
The strategy of introducing cysteine residues
throughout the cavity-lining helices has yielded consid-
erable, but often contradictory, mechanistic informa-
tion on helical involvement in ABCB1 function. One
approach has been to ascertain the propensity of the
introduced cysteine to react with a maleimide–drug
(i.e. thiol reactive) conjugate [14,28]. Covalent
attachment of the drug conjugate was reasoned to
demonstrate that the residue was located within the
drug-binding pocket. The data were supported by the
prevention of cysteine attachment using a drug without

the maleimide moiety. Other investigations have
focused on the consequences of the cysteine mutation
per se, or following attachment of thiol-reactive, non-
substrate, probe molecules [15,29,30]. This approach
also allowed investigation of the helical topography
and how this alters during functionally relevant
conformational changes in the protein.
The studies discussed above have been ongoing for
several years and involve many independent research
teams. It appears that a large number of helices (TM1,
TM4-7 and TM9-12) may line the central cavity, con-
tribute to the drug-binding pocket and ⁄ or regulate
conformational coupling within ABCB1. It is worth
noting that TM6 and TM12 have consistently been
implicated in important functional roles for ABCB1.
The number of TM helices (9 of 12) predicted to line
the central cavity suggests a large dimension. Not all
residues within the cavity-lining helices demonstrate a
strong functional role and furthermore, certain
mutations show selectivity towards different substrate
molecules. A central, often aqueous filled, cavity is not
limited to ABCB1 because structural studies with a
number of ABC transporters have revealed similar
findings [17,31,32].
Collectively, these data may be consistent with a
large binding domain or pocket and the presence of a
drug imparting distinct conformational alterations akin
to the ‘induced fit’ model for drug binding.
The domain interface model
As is the case for all ABC transport proteins, the

membrane-spanning region of ABCB1 comprises two
domains. Both halves of the TMD appear to be func-
tionally important and capable of drug interaction, a
point that is strongly supported by studies that photo-
label ABCB1, digest the protein and identify fragments
containing the attached drug (Table 1). The electron
microscopy structures for ABCB1 display a discontinu-
ity in the TMD region; however, resolution of the data
does not yet enable prediction of the proximal helices.
A number of cross-linking studies have focused on
generating a spatial topology map for ABCB1 and a
similar aim has been targeted through molecular mod-
els based on the high-resolution structures of Sav186
and MsbA [33–36]. The consensus appears to be that
one of the domain interfaces is mediated by TM5 ⁄ 8,
whereas the other comprises TM11 ⁄ 3 with TM2 poten-
tially contributing.
Cysteines introduced at these two interfacial regions
demonstrated avid accessibility to conjugation with
drug–maleimide compounds, suggesting an involve-
ment in drug binding. However, addition of drug with-
out maleimide did not offer protection against
chemical modification. Perhaps the most significant
support for the interfacial region comprising a drug-
binding site was obtained using the photoactive propa-
fenone derivative [
3
H]GPV51 [8]. Following labelling
and chemical digestion, the fragments were analysed
for the presence of drug using MALDI-TOF MS. This

powerful approach also provided data on the labelling
density and indicated that fragments from TM3, TM5,
Table 1. (Continued )
Date Reference Comments
2007 [15,16,30] Strategy: Cysteine-directed mutagenesis of TM6
Result: Mutations did not alter initial drug binding however the helix was crucial in mediating TMD–NBD
communication
2009 [33] Strategy: MsbA-based model to dock drugs onto
Result: Proposed a number of binding clusters dotted throughout the TMDs and that multiple drugs could
interact simultaneously
E. Crowley and R. Callaghan Drug binding – gates or cavity?
FEBS Journal 277 (2010) 530–539 ª 2009 The Authors Journal compilation ª 2009 FEBS 533
TM8 and TM11 accounted for > 70% of the total
bound drug. Based on homology modelling and cross-
linking data, it was reasoned that this labelling
occurred in two pockets comprising TM5 ⁄ 8 and
TM3 ⁄ 11, and that these provided the interface between
the N- and C-terminal halves of the TMDs.
How convincing are the data used to
identify the binding sites?
Considerable debate on the merits and applicability of
the two models continues to rage and remains unre-
solved. Is the experimental data stronger for either
model and are the models mutually exclusive? Indeed,
there are a number of issues with the supporting data
for both models. More correctly, it is interpretation of
the supporting data that requires further consideration
or refinement. This section highlights many of the
drawbacks or limitations of the data currently in use
to locate the drug-binding sites on ABCB1.

The resistance profile of cells expressing ABCB1 is a
frequently used reporter of activity and has been
employed to infer the functional consequences of
mutations in the protein [10,12]. However, cytotoxicity
assays inform on a whole phenotype and this often
means multiple mechanisms of resistance, particularly
in the case of cell lines selected in the presence of high
anticancer drug concentrations. The precise quantita-
tive contribution of ABCB1 to the phenotype is diffi-
cult to assess in such a complex system. Other
functional assays used in whole cells or proteolipo-
somes include steady-state drug accumulation or stim-
ulation of ATP hydrolysis [37–39]. Drug transport is a
multistep process involving drug binding, ATP hydro-
lysis and conformational changes leading to trans-
bilayer movement. Therefore, attributing altered levels
of transport specifically to the initial drug-binding step
is difficult to do with conviction. ATPase activity and
its stimulation or inhibition by drugs are also complex,
involving considerably more than simply drug binding.
Perhaps the most conclusive data are provided by
directly measuring the drug–ABCB1 interaction using
equilibrium binding assays or photoaffinity labelling
procedures [1,3,40]. Moreover, the greatest confidence
may only be afforded by extensive dose–response
analyses to measure capacity and affinity changes in
drug binding, rather than reliance on a single drug
concentration.
Often the pharmacology assays examining ABCB1
function rely on modified versions of the drug; for

example, photoactive azide derivatives or drug–malei-
mide conjugates. Drug derivatives require that the
active moiety (e.g. azide) does not interfere with the
‘normal’ drug–protein interaction. In other words, they
must lie outside the actual drug pharmacophore other-
wise the true binding affinity or process is not being
examined. Another drawback of these derivatives is
that the active moiety may exhibit considerable
motion. This issue was validated for the interaction of
azidopine with l-type calcium channels, wherein the
azide moiety exhibited a conical range of motion from
the point of contact with the protein [41]. Conse-
quently, the region labelled on the protein may be
distinct from the actual binding site or be located at
multiple sites.
Many strategies to locate the binding sites involve
digestion of ABCB1 following labelling with reactive
drugs. Unfortunately, neither chemical nor proteolytic
digestion is complete and therefore frequently displays
a heterogeneous fragmentation pattern. Reproducibil-
ity issues have also been noted and the combination
renders the identification of fragments a difficult task.
Early attempts favoured the use of antibodies to iden-
tify drug-containing fragments, but generating antibod-
ies for all the fragments produced is an unlikely
scenario, particularly for shorter fragments [5–7]. The
recent work of Pleban et al. [8] developed a MALDI-
TOF MS approach to circumvent these issues and it
certainly warrants greater usage.
ABCB1 adopts numerous stable conformations with

the impetus for most of the transitions caused by
nucleotide binding ⁄ hydrolysis and drug binding. Con-
sequently, labelling or binding conditions need to be
carefully controlled to prevent the final data represent-
ing an ‘averaged’ and potentially noninitial conforma-
tion. For example, cross-linking between helices in the
presence or absence of drug substrates is sensitive to
protein movement within the TMDs and may reflect
allosteric changes rather than simple steric inhibition
of cross-linkage. Similarly, protection assays using
unlabelled compound (see previous section) may be
affected. The labelled and unlabelled drugs could con-
ceivably bind at distinct sites and the latter may simply
cause a conformational change in ABCB1 that alters
the accessibility of the target cysteine residue.
Molecular modelling approaches in the analysis of
ABCB1 are currently based on non-ABCB1 structures
and therefore a degree of caution is prudent [33–36].
Two principle structures are employed, namely MsbA
and Sav186, but unfortunately these proteins display
considerable differences. MsbA, particularly in the basal
configuration, has large separation between the two
monomers and there is considerable debate on the phys-
iological significance of the various conformations. In
comparison, Sav186 displays considerable domain
swapping between the two monomers, although this
Drug binding – gates or cavity? E. Crowley and R. Callaghan
534 FEBS Journal 277 (2010) 530–539 ª 2009 The Authors Journal compilation ª 2009 FEBS
finding has not yet been validated biochemically. Are
either or both structures correct or do they represent

crystal artefacts? More importantly in respect to this
minireview, how similar is the structure of ABCB1?
Until such discrepancies are reconciled, interpretation of
these models can only be considered speculative. Several
attempts at docking drugs onto the models have been
attempted [33,35,36]. However, without absolute knowl-
edge of the drug-binding site location or the features of
the drug–ABCB1 interaction being fully described, the
data from modelling approaches also need careful and
cautious interpretation.
Recently, the structure of mouse ABCB1 has been
obtained using X-ray crystallography and although
this is a significant breakthrough, structural resolution
was at 3.8 A
˚
in the absence of drug. Structural infor-
mation was also obtained by ‘soaking’ crystals in the
presence of an ABCB1 inhibitor and the resolution
obtained was slightly lower at 4.4 A
˚
. The cyclic hexa-
peptide inhibitor was synthesized specifically for this
study and its relationship to the established pharmaco-
logical drug interactions sites (e.g. site I for vinblas-
tine) is presently unclear. In addition, the current level
of resolution precludes atomic detail on the drug–
protein interaction. Many of the residues implicated in
binding to the custom-built inhibitor are, however,
equivalent to those from the biochemical investiga-
tions. A great deal more analysis and functional inves-

tigation based on this structure are required and
presumably underway. As the resolution undoubtedly
improves towards, or better than, 3 A
˚
so too will our
understanding of the molecular interaction of drugs
with ABCB1. In particular, we need information on
the forces mediating drug binding (e.g. hydrogen bond-
ing), the local solute environment (e.g. pH) and the
dimensions or topography of the binding site.
Can the data be reconciled into a map
of the sites?
Where do we stand on the issue of the location of
drug-binding sites on ABCB1? There is clearly a
wealth of information implicating several regions of
the protein (Fig. 1). However, the previous section
appears to cast doubt on the findings. This is not
meant to be the case, but simply to urge some degree
of caution and care in data interpretation. Figure 1
also highlights the fact that a considerable proportion
of the protein is involved in drug binding and ⁄ or medi-
ating communication pathways between the TMDs
and the NBDs. This section aims to reconcile the data
into a working model of drug interaction with
ABCB1.
Overall, the data and observations are weighted
towards (but not exclusively) three pairs of helices
(TM3 ⁄ 11, TM5 ⁄ 8 and TM6 ⁄ 12) playing a significant
role in drug binding. The TM6 ⁄ 12 pair is clearly
involved in the translocation process, but its precise

role is not yet fully resolved. Mutations in these helices
alter cytotoxicity profiles, overall drug accumulation
and ATP hydrolysis. The controversy relates to the
involvement in drug binding per se. Studies with drug–
maleimide conjugates favour a role, whereas recent
1 2 3 4 5 6
7
8
9 10 11
12
NBD1
NBD2
Fig. 1. Topological map of the regions of ABCB1 implicated in drug
binding. Schematic depiction of the topological organization of
ABCB1 with the rectangular TM helices and circular NBDs. Areas
shaded in light green indicate regions of the protein thought to
mediate drug binding. The deeper shade of green indicates a
greater amount of observational data supporting this role. Pink
shading reveals areas of the protein thought to mediate communi-
cation pathways involved in the translocation process.
11
3
12
8
6
5
11
12
8
6

5
d
1
1
3
12
8
6
5
3
d
d
d
11
3
12
8
6
5
ATP
binding
ATP
hydrolysis
Pi
dissociation
Fig. 2. The two-step model of drug translocation by ABCB1. (Top
left) An arrangement of TM helices within the membrane-spanning
domain of ABCB1. Numbered circles refer to specific helices and
only a small selection are shown. The brown star-shaped object
refers to a typical transported substrate of the protein. Altered col-

ouration of the TM helices indicates that the segment has under-
gone a conformational change. A full description of the initial
binding of drug to the high-affinity binding sites on ABCB1 and the
subsequent shift to a low-affinity site and a final dissociation to
complete translocation across the membrane is given in the text.
E. Crowley and R. Callaghan Drug binding – gates or cavity?
FEBS Journal 277 (2010) 530–539 ª 2009 The Authors Journal compilation ª 2009 FEBS 535
work using radiolabelled substrates suggests they are
not involved. The working model of drug binding to
ABCB1 (Fig. 2) will assume that they do not mediate
drug binding, at least only the initial high-affinity step.
They do, however, propagate TMD M NBD commu-
nication essential to coupling the translocation process.
This model is based on (but not limited to) the hydro-
lysis of a single nucleotide per translocation event, as
suggested by a number of excellent biochemical studies
[42–44]. Moreover, the reader is directed to an earlier
review for a more exhaustive mechanistic model on the
complex sequence of events within the translocation
mechanism of ABCB1 [45].
The other two helical pairs (TM3 ⁄ 11 and TM5 ⁄ 8)
have been identified as binding drug using maleimide–
drug conjugates, photo-cross-linking, mutagenesis and
molecular modelling. Consequently, these helical pairs
have been assigned as the initial drug-binding sites in
the working model. Modelling and cross-linking data
place these helices at the domain interface between the
N- and C-terminal halves of the TMD. Drugs may
bind to either TM3 ⁄ 11 or TM5 ⁄ 8 depending on their
physicochemical properties, given the pharmacological

data indicating multiple distinct sites in ABCB1.
The binding of ATP and the subsequent dimeriza-
tion of the NBDs are believed to instigate the switch
of the drug-binding sites from high to low affinity; i.e.
the so-called ‘power stroke’ [46–50]. A recent article
[51] further supports the data produced by Martin
et al. that provided the original underlying evidence
for a shift in the drug-binding site affinity in response
to ATP binding at the NBDs [48]. The stimulus for
this switch in ABCB1 is propagated through the
TMDs via conformational changes in TM6 ⁄ 12, given
their effects on the transport process and their direct
contact with the NBDs. The consequence of this
switch is that the drug now enters the central cavity.
At this point, the TM helices lining the cavity (includ-
ing TM6 and TM12) make contact with the bound
drug, albeit with low affinity. This two-stage binding
model therefore takes into account data implicating
both the central cavity and domain interface models.
Further conformational changes caused by progres-
sion of the catalytic cycle result in the dissociation of
drug from the central cavity and the final restoration
of ABCB1 to its initial transport ready conformation.
This model obviously requires further validation.
However, it partially reconciles a substantial number
of the available biochemical observations. In addi-
tion, it does not contravene the large number of
studies that have described important characteristics
of the drug-binding sites and their coupling to the
NBDs.

How can we fully validate the two-step binding
model? More biochemical data verifying the sites are
of obvious importance, in particular, the cross-linking
of drugs with subsequent digestion and MS analysis
appears a powerful strategy. Structural data at high
resolution (3 A
˚
or greater) would also be of invaluable
assistance. Generating structural data in the presence
of multiple drugs and protein in different conforma-
tions would provide the ideal information to fully elu-
cidate the issue of drug binding to ABCB1. Such a
goal remains elusive, but is clearly achievable given the
recent published structure. Until then, however, the
debate on the precise location of drug-binding sites in
the protein will continue to rumble.
Acknowledgements
This investigation was generously supported by a
Cancer Research UK Studentship to Emily Crowley
(C362 ⁄ A5502) for which Richard Callaghan was the
principal investigator.
References
1 Ferry DR, Russell MA & Cullen MH (1992) P-Glyco-
protein possesses a 1,4-dihydropyridine selective drug
acceptor site which is allosterically coupled to a vinca
alkaloid selective binding site. Biochem Biophys Res
Commun 188, 440–445.
2 Lugo MR & Sharom FJ (2005) Interaction of LDS-751
and rhodamine 123 with P-glycoprotein: evidence for
simultaneous binding of both drugs. Biochemistry 44,

14020–14029.
3 Martin C, Berridge G, Higgins CF, Mistry P, Charlton
P & Callaghan R (2000) Communication between multi-
ple drug binding sites on P-glycoprotein. Mol Pharma-
col 58, 624–632.
4 Orlowski S, Mir LM, Belehradek J & Garrigos M
(1996) Effects of steroids and verapamil on P-glycopro-
tein ATPase activity: progesterone, desoxycorticosterone
and verapamil are mutually non-exclusive modulators.
Biochem J 317, 515–522.
5 Bruggemann EP, Germann UA, Gottesman MM &
Pastan I (1989) Two different regions of P-glycoprotein
[corrected] are photoaffinity-labeled by azidopine. J Biol
Chem 264, 15483–15488.
6 Greenberger LM (1993) Major photoaffinity labeling
sites for iodoaryl azidoprazosin in P-glycoprotein are
within or immediately C-terminal to transmembrane
domains 6 and 12. J Biol Chem 268, 11417–11425.
7 Greenberger LM, Lisanti CJ, Silva JT & Horwitz SB
(1991) Domain mapping of the photoaffinity drug bind-
ing sites in P-glycoprotein encoded by mouse mdr1b.
J Biol Chem 266, 20744–20751.
Drug binding – gates or cavity? E. Crowley and R. Callaghan
536 FEBS Journal 277 (2010) 530–539 ª 2009 The Authors Journal compilation ª 2009 FEBS
8 Pleban K, Kopp S, Csaszar E, Peer M, Hrebicek T,
Rizzi A, Ecker GF & Chiba P (2005) P-Glycoprotein
substrate binding domains are located at the transmem-
brane domain ⁄ transmembrane domain interfaces: a
combined photoaffinity labelling-protein homology
modeling approach. Mol Pharmacol 67, 365–374.

9 Devine SE, Ling V & Melera PW (1992) Amino acid
substitutions in the sixth transmembrane domain of
P-glycoprotein alter multidrug resistance. Proc Natl
Acad Sci USA 89 , 4564–4568.
10 Kajiji S, Talbot F, Grizzuti K, Van Dyke-Phillips V,
Agresti M, Safa AR & Gros P (1993) Functional analy-
sis of P-glycoprotein mutants identifies predicted trans-
membrane domain 11 as a putative drug binding site.
Biochemistry 32, 4185–4194.
11 Loo TW & Clarke DM (1993) Functional consequences
of phenylalanine mutations in the predicted transmem-
brane domain of P-glycoprotein. J Biol Chem 268,
19965–19972.
12 Ma JF, Grant G & Melera PW (1997) Mutations in the
sixth transmembrane domain of P-glycoprotein that
alter the pattern of cross-resistance also alter sensitivity
to cyclosporin A reversal. Mol Pharmacol 51, 922–930.
13 Loo TW & Clarke DM (2001) Determining the dimen-
sions of the drug-binding domain of human P-glycopro-
tein using thiol cross-linking compounds as molecular
rulers. J Biol Chem 276, 36877–36880.
14 Loo TW & Clarke DM (2001) Defining the drug-bind-
ing site in the human multidrug resistance P-glycoyco-
protein using a methanethiosulfonate analog of
verapamil, MTS-verapamil. J Biol Chem 276, 14972–
14979.
15 Rothnie A, Storm J, Campbell J, Linton KJ, Kerr ID
& Callaghan R (2004) The topography of transmem-
brane segment six is altered during the catalytic cycle of
P-glycoprotein. J Biol Chem 279, 34913–34921.

16 Storm J, O’Mara ML, Crowley EH, Peall J, Tieleman
DP, Kerr ID & Callaghan R (2007) Residue G346 in
transmembrane segment six is involved in inter-domain
communication in P-glycoprotein. Biochemistry 46,
9899–9910.
17 Dawson RJ & Locher KP (2007) Structure of the multi-
drug ABC transporter Sav1866 from Staphylococ-
cus aureus in complex with AMP–PNP. FEBS Lett 581,
935–938.
18 Ward A, Reyes CL, Yu J, Roth CB & Chang G (2007)
Flexibility in the ABC transporter MsbA: alternating
access with a twist. Proc Natl Acad Sci USA 104,
19005–19010.
19 Khare D, Oldham ML, Orelle C, Davidson AL & Chen
J (2009) Alternating access in maltose transporter medi-
ated by rigid-body rotations. Mol Cell 33, 528–536.
20 Lee J-Y, Urbatsch IL, Senior AE & Wilkens S (2002)
Projection structure of P-glycoprotein by electron
microscopy. Evidence for a closed conformation of the
nucleotide binding domains. J Biol Chem 277, 40125–
40131.
21 Lee J-Y, Urbatsch IL, Senior AE & Wilkens S (2008)
Nucleotide-induced structural changes in P-glycoprotein
observed by electron microscopy. J Biol Chem 283,
5769–5779.
22 Rosenberg MF, Callaghan R, Ford RC & Higgins CF
(1997) Structure of the multidrug resistance P-glycopro-
tein to 2.5 nm resolution determined by electron
microscopy and image analysis. J Biol Chem 272,
10685–10694.

23 Rosenberg MF, Kamis AB, Callaghan R, Higgins CF
& Ford RC (2003) Three-dimensional structures of the
mammalian multidrug resistance P-glycoycoprotein
demonstrate major conformational changes in the trans-
membrane domains upon nucleotide binding. J Biol
Chem 278, 8294–8299.
24 Rosenberg MF, Velarde G, Ford RC, Martin C, Ber-
ridge G, Kerr ID, Callaghan R, Schmidlin A, Wooding
C, Linton KJ et al. (2001) Repacking of the transmem-
brane domains of P-glycoprotein during the transport
ATPase cycle. EMBO J 20, 5615–5625.
25 Loo TW & Clarke DM (2005) Recent progress in
understanding the mechanism of P-glycoprotein-medi-
ated drug efflux. J Membr Biol 206, 173–185.
26 Loo TW, Bartlett MC & Clarke DM (2004) Disulfide
cross-linking analysis shows that transmembrane seg-
ments 5 and 8 of human P-glycoprotein are close
together on the cytoplasmic side of the membrane.
J Biol Chem 279, 7692–7697.
27 Loo TW & Clarke DM (2000) Identification of residues
within the drug binding domain of the human multi-
drug resistance P-glycoprotein by cysteine-scanning
mutagenesis and reaction with dibromobimane. J Biol
Chem 275, 39272–39278.
28 Loo TW, Bartlett MC & Clarke DM (2003) Methan-
ethiosulfonate derivatives of rhodamine and verapamil
activate human P-glycoprotein at different sites. J Biol
Chem 278, 50136–50141.
29 Crowley E, O’Mara ML, Reynolds C, Tieleman DP,
Storm J, Kerr ID & Callaghan R (2009) Transmem-

brane helix 12 modulates progression of the ATP cata-
lytic cycle in ABCB1. Biochemistry 48, 6249–6258.
30 Storm J, Modok S, O’Mara ML, Tieleman DP, Kerr
ID & Callaghan R (2008) Cytosolic region of TM6 in
P-glycoprotein: topographical analysis and functional
perturbation by site directed labeling. Biochemistry 47,
3615–3624.
31 Locher KP, Lee AT & Rees DC (2002) The E. coli
BtuCD structure: a framework for ABC transporter
architecture and mechanism. Science 296, 1091–1098.
32 Oldham ML, Khare D, Quiocho FA, Davidson AL
& Chen J (2007) Crystal structure of a catalytic inter-
mediate of the maltose transporter. Nature 450, 515–
521.
E. Crowley and R. Callaghan Drug binding – gates or cavity?
FEBS Journal 277 (2010) 530–539 ª 2009 The Authors Journal compilation ª 2009 FEBS 537
33 Becker JP, Depret G, Van Bambeke F, Tulkens PM &
Prevost M (2009) Molecular models of human P-glyco-
protein in two different catalytic states. BMC Struct
Biol 9, 3, doi:10.1186/1472-6807-9-3.
34 O’Mara ML & Tieleman DP (2007) P-Glycoprotein
models of the apo and ATP-bound states based on
homology with Sav1866 and MalK. FEBS Lett 581,
4217–4222.
35 Ravna AW, Sylte I & Sager G (2007) Molecular model
of the outward facing state of the human P-glycoprotein
(ABCB1), and comparison to a model of the human
MRP5 (ABCC5). Theor Biol Med Model 4, 33,
doi:10.1186/1742-4682-4-33.
36 Vandevuer S, Van Bambeke F, Tulkens PM & Prevost

M (2006) Predicting the three-dimensional structure of
human P-glycoprotein in absence of ATP by computa-
tional techniques embodying crosslinking data: insight
into the mechanism of ligand migration and binding
sites. Proteins Struct Funct Bioinform 63, 466–478.
37 Al-Shawi MK & Senior AE (1993) Characterization of
the adenosine triphosphatase activity of Chinese ham-
ster P-glycoprotein. J Biol Chem 268, 4197–4206.
38 Martin C, Berridge G, Mistry P, Higgins C, Charlton P
& Callaghan R (1999) The molecular interaction of the
high affinity reversal agent XR9576 with P-glycoprotein.
Br J Pharmacol 128, 403–411.
39 Sharom FJ (1995) Characterization and functional
reconstitution of the multidrug transporter. J Bioenerg
Biomembr 27, 15–22.
40 Safa AR, Stern RK, Choi K, Agresti M, Tamai I,
Mehta ND & Roninson IB (1990) Molecular basis of
preferential resistance to colchicine in multidrug resis-
tant human cells conferred by Gly185-Val185 substitu-
tion in P-glycoprotein. Proc Natl Acad Sci USA 87,
7225–7229.
41 Glossmann H, Ferry DR, Striessnig J, Goll A & Moos-
burger K (1987) Resolving the structure of the Ca
2+
channel by photoaffinity labeling. Trends Pharmacol Sci
8, 95–100.
42 Carrier I, Julien M & Gros P (2003) Analysis of cata-
lytic carboxylate mutants E552Q and E1197Q suggests
asymmetric ATP hydrolysis by the two nucleotide-bind-
ing domains of P-glycoprotein. Biochemistry 42, 12875–

12885.
43 Urbatsch IL, Sankaran B, Weber J & Senior AE (1995)
P-Glycoprotein is stably inhibited by vanadate-induced
trapping of nucleotide at a single catalytic site. J Biol
Chem 270, 19383–19390.
44 Urbatsch IL, Tyndall GA, Tombline G & Senior AE
(2003) P-Glycoprotein catalytic mechanism: studies of
the ADP-vanadate inhibited state. J Biol Chem 278,
23171–23179.
45 Callaghan R, Ford RC & Kerr ID (2006) The translo-
cation mechanism of P-glycoprotein. FEBS Lett 580,
1056–1063.
46 Higgins CF & Linton KJ (2004) The ATP switch model
for ABC transporters. Nat Struct Mol Biol 11, 918–926.
47 Maki N, Moitra K, Ghosh P & Dey S (2006) Allosteric
modulation bypasses the requirement for ATP hydroly-
sis in regenerating low affinity transition state confor-
mation of human P-glycoprotein. J Biol Chem 281,
10769–10777.
48 Martin C, Higgins CF & Callaghan R (2001) The vin-
blastine binding site adopts high- and low-affinity con-
formations during a transport cycle of P-glycoprotein.
Biochemistry 40, 15733–15742.
49 Sauna ZE, Nandigama K & Ambudkar SV (2006)
Exploiting reaction intermediates of the ATPase reac-
tion to elucidate the mechanism of transport by P-gly-
coprotein (ABCB1). J Biol Chem 281, 26501–26511.
50 Abele R & Tampe R (2004) The ABCs of immunology:
structure and function of TAP, the transporter associ-
ated with antigen processing. Physiology (Bethesda) 19,

216–224.
51 Aanismaa P, Gatlik-Landwojtowicz E & Seelig A
(2008) P-Glycoprotein senses its substrates and the lat-
eral membrane packing density: consequences for the
catalytic cycle. Biochemistry 47, 10197–10207.
52 Greenberger LM, Yang C-PH, Gindin E & Horwitz SB
(1990) Photoaffinity probes for the a
1
-adrenergic recep-
tor and the calcium channel bind to a common domain
in P-glycoprotein. J Biol Chem 265, 4394–4401.
53 Pawagi AB, Wang J, Silverman M, Reithmeier RA &
Deber CM (1994) Transmembrane aromatic amino acid
distribution in P-glycoprotein. A functional role in
broad substrate specificity. J Mol Biol 235 , 554–564.
54 Zhang X, Collins KI & Greenberger LM (1995) Func-
tional evidence that transmembrane 12 and the loop
between transmembrane 11 and 12 form part of the
drug-binding domain in P-glycoycoprotein encoded by
MDR1. J Biol Chem 270, 5441–5448.
55 Dey S, Ramachandra M, Pastan I, Gottesman MM &
Ambudkar SV (1997) Evidence for two nonidentical
drug-interaction sites in the human P-glycoycoprotein.
Proc Natl Acad Sci USA 94, 10594–10599.
56 Seelig A (1998) A general pattern for substrate
recognition by P-glycoprotein. Eur J Biochem 251, 252–
261.
57 Hafkemeyer P, Dey S, Ambudkar SV, Hrycyna CA,
Pastan I & Gottesman MM (1998) Contribution to sub-
strate specificity and transport of nonconserved residues

in transmembrane domain 12 of human P-glycoprotein.
Biochemistry 37, 16400–16409.
58 Isenberg B, Thole H, Tummler B & Demmer A (2001)
Identification and localization of three photobinding
sites of iodoarylazidoprazosin in hamster P-glycopro-
tein. Eur J Biochem 268, 2629–2634.
59 Song J & Melera PW (2001) Transmembrane domain
(TM) 9 represents a novel site in P-glycoprotein that
affects drug resistance and cooperates with TM6 to
Drug binding – gates or cavity? E. Crowley and R. Callaghan
538 FEBS Journal 277 (2010) 530–539 ª 2009 The Authors Journal compilation ª 2009 FEBS
mediate [
125
I]iodoarylazidoprazosin labeling. Mol
Pharmacol 60, 254–261.
60 Lugo MR & Sharom FJ (2005) Interaction of LDS-751
with P-glycoprotein and mapping of the location of the
R drug binding site. Biochemistry 44, 643–655.
61 Loo TW & Clarke DM (2005) Do drug substrates enter
the common drug-binding pocket of P-glycoprotein
through ‘gates’? Biochem Biophys Res Commun 329,
419–422.
62 Loo TW, Bartlett MC & Clarke DM (2006) Trans-
membrane segment 7 of human P-glycoprotein forms
part of the drug-binding pocket. Biochem J 399, 351–
359.
63 Loo TW, Bartlett MC & Clarke DM (2006) Trans-
membrane segment 1 of human P-glycoprotein con-
tributes to the drug-binding pocket. Biochem J 396,
537–545.

64 Omote H & Al-Shawi MK (2006) Interaction of trans-
ported drugs with the lipid bilayer and P-glycoprotein
through a solvation exchange mechanism. Biophys J 90,
4046–4059.
E. Crowley and R. Callaghan Drug binding – gates or cavity?
FEBS Journal 277 (2010) 530–539 ª 2009 The Authors Journal compilation ª 2009 FEBS 539